The synthesis of polypeptides by conventional solid phase methods, amino acid by amino acid, is limited by low yields when the polypeptides synthesized are large in size. In order to overcome this limitation, it is known to assemble two polypeptides by chemical ligation in order to produce a longer polypeptide.
The total synthesis of polypeptides is increasingly useful for the preparation of proteins with well-defined structures. The chemical ligation methods provide a response to this need, however they prove limited in their use and their industrial application.
Generally, in these methods, it is desired that the bond between the polypeptides assembled by ligation is native, i.e. corresponds to the natural structure of the polypeptides.
The main native ligation method currently existing is that of Kent and Dawson, described for example in the international applications WO 96/34878 and WO 98/28434. This method is based on a chemoselective reaction between a (C-terminal) thioester peptide and a cysteinyl-peptide. Nevertheless, the main drawback of this method is the production of thioester peptides which requires complex chemical processes. In particular, according to an assembly method described by S. Kent (Kinetically Controlled Ligation (KCL)), it is necessary to synthesize the A-SAr, H-Cys-B-SAlk, and H-Cys-C fragments. However A-SAr type fragments are difficult to produce and prove to be susceptible to hydrolysis. Furthermore, this method does not make it possible to go beyond the assembly of 3 fragments.
An alternative method is the so-called Staudinger ligation, described in the international applications WO 01/68565 and WO 01/87920. This comprises the reaction of a phosphinothioester with an azide and the hydrolysis of the combined reagents to form an amide bond. However this method is difficult to apply on an industrial scale.
Another method, described in the international application WO 2007/037812, is based on the reaction of an α-keto acid with an alkoxyamine in a decarboxylative condensation reaction. However, the keto acids are molecules which are difficult to produce and to incorporate into peptides. Also, this third method is difficult to apply in peptide synthesis laboratories that do not have the means for carrying out complex organic syntheses.
The publication by D. Bang, B. L. Pentelute and S. B. H. Kent, Angew. Chem. Int. Ed. 2006, 45, 3985-3988 proposes a synthesis route involving peptide-(thiophenylesters) with Cys-peptide-thioesters, and the publication by W. Hou et al., Org Lett., (2010), 22 Dec. 2010, proposes the formation of peptide-thioesters for the synthesis of proteins. However these methods cannot prevent competition between the reactions of the different thioesters, inevitably leading to mixtures that can be difficult to separate, therefore affecting the purity of the final product obtained, and to inevitable losses of yield.
Finally, the publication by O. Melnyk et al., Org. Lett., 12(22), 5238-41 (2010) describes the native ligation of peptides by means of peptide-bis(sulphanylethyl)amino fragments. However this method has never to date been used for the synthesis of peptides with multiple fragments.
The transfer to the industrial scale of methods making it possible to implement peptide syntheses by total synthesis is a need that requires methods to be found which are simple, inexpensive, producing quality products of high purity which satisfy industrial health requirements.
For the abovementioned reasons, it has become essential to find a total synthesis method, which is coherent and can be used on an industrial scale, making it possible to synthesize a peptide chain formation of the desired length and nature. Particularly a method involving assembly from the N-terminal end towards the C-terminal end, which offers qualities of simplicity of production and purity of the peptides or polypeptides obtained.